PAPER ELECTROPHORESIS AS A
QUANTITATIVE METHOD: MEASUREMENT OF
ALPHA AND BETA LIPOPROTEIN
CHOLESTEROL
Thomas A. Langan, … , E. L. Durrum, William P. Jencks
J Clin Invest. 1955;34(9):1427-1436. https://doi.org/10.1172/JCI103192.
Research Article
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PAPER ELECTROPHOREtSIS AS A QUANTITATIVE METHOD:
MEASUREMENT OF ALPHA AND BETA LIPOPROTEIN
CHOLESTEROL
By THOMAS A. LANGAN, E. L. DURRUM,l AND WILLIAM P. JENCKS2
(From the Department of Pharmacology, Army Medical Service Graduate School, Walter Reed Army Medical Center, Washington, D. C.)
(Submitted for publication March 21, 1955; accepted May 11, 1955)
The development of methods for the separation and measurement of serum lipoproteins by ultra-centrifugal flotation, fractional precipitation and
zone electrophoresis has made possible studies of the metabolism and pathological significance of several lipoprotein fractions. Except for early studies by Lindgren, Elliott, Gofman, Jones, Lyon, and Strisower on the relationship of beta lipopro-teinsubfractions of certain flotation rates to athero-sclerosis (1, 2) most of these studies have been concerned with alpha and beta lipoproteins, as de-fined by density in the ultracentrifugal techniques
(3-5), by solubility in the chemical fractionation
techniques (6) and by mobility in electrophoretic separation. Using a protein fractionation proce-duredeveloped by Cohn and his group (7), Barr, Russ, and Eder (8, 9) found that in 33 athero-sclerotic individuals an average of only 13.6 per
cent of the total serum cholesterol was associated withthe alpha lipoprotein fraction as compared to 27.8 per cent in 85 normals.
In an attempt to avoid the complex equipment and technique and the large amount of serum re-quired for the ultracentrifugal and chemical tech-niques, several workers have taken advantage of the ease of separation of alpha and beta lipopro-teins by electrophoresis in anti-convection media such as starch and filter paper. The separated lipoproteins were then measured either by stain-ing techniques, which are dependent on the
solu-bility of lipid dyes in certain serum lipids (10-14
and others), or by the more specific method of elution and chemical determination of the choles-terol in the alpha and beta lipoprotein fractions
(15-21). Thus Nikkila (15), using 0.24 ml. of
1Present address: Department of Pharmacology,
Stan-ford University Medical School, Clay & Webster Sts.,
San Francisco 15, California.
2Present address: Biochemical Research Laboratory,
Massachusetts General Hospital, Fruit St., Boston, Mass.
serum separated on three sheets of 11 x 30 -cm. paper has shown a decrease in alpha lipoprotein cholesterol in atherosclerotic patients, and Kunkel and Slater (22) have made use of starch blocks
assupporting media in order toavoid the "tailing" oflipoproteins encountered with filter paper. The techniques of cholesterol measurement used in these studies have thedisadvantages ofbeing
time-consuming and of requiring larger amounts of serum than can be adequately separated on a
single paper strip of reasonable size.
Zlatkis, Zak, and Boyle have recently described
a method for the determination of the total cho-lesterol of whole serum without extraction,
pre-cipitation or hydrolysis, which is several times more sensitive than the Schoenheimer-Sperry
technique and which gives equal color with cho-lesterol and cholesterol esters (23). Use of this method on whole serum has been criticized by Best, Van Loon, Wathem, and Seger (24) who found that it gave high values compared to the
Schoenheimer-Sperry method, and by Fiirst and Lange (25) who found variations in color with changes intemperature or amounts of reagent.
A method is described here for the
determina-tion of alpha and beta lipoprotein cholesterol on
eluates from 0.02 ml. of electrophoretically sepa-rated serum. Cholesterol is determined by a
modification of the Zlatkis, Zak, and Boyle pro-cedure which gives satisfactory agreement with the Schoenheimer-Sperry technique. The small amount of serumrequired allows a clearcut
sepa-ration of the alpha and beta fractions in a short
migration distance without overloading of the
paper and without measurable error due to "tail-ing." The method is suited for large scale
clini-cal studies with regard to time, technique and ap-paratus involved and meets clinical standards of reproducibility and quantitative recovery.
MATERIALS AND METHODS
Reagents
1. Barbital buffer, pH 8.6, 0.05,. 1.84 g. of
diethyl-barbituric acid and 10.3 g. of sodium barbital were
dis-solved inone liter of distilled water.
2. Sudan black staining solutiont was prepared
ac-cording to Swahn (10). One liter of 60per cent ethanol was saturated with 1 g. of Sudan black B (National
Aniline Division, Allied Chemical and Dye Corporation) by bringing to the boiling point. The solution was
cooled to room temperature, filtered, and stored in a
closed container.
3. Chloroformt-methanol solvent: Two volumes of
re-distilled chloroform were mixed with one volume of re-distilled methanol just prior touse. These solvents were
freshly distilled no more than two weeks before use.
4. Standard cholesterol soluitiont: One hundred mg. of
recrystallized cholesterol was dissolved in glacial acetic
acid and made up to 100 ml. at 250 C. This solution was stored frozen in the refrigerator and warmed to 240 to 260 C. before samples were withdrawn. Under these conditions no measurable deterioration of the standard
solution was found over a period of four months.
5. Glacial acetic acid: Except as otherwise specified,
reagent grade glacial acetic acid (Fisher Chemical Co.)
was used. Acetic acid was tested for the presence of
glyoxylic acid which would give color with tryptophane by a modification of the method of Brice (26). Five
hundredths ml. of 100 mg. per cent aqueous tryptophane was added to 0.95 ml. of the acetic acid to be tested. A volume of 0.65 ml. of sulfuric acid was then added, and
the mixture was heated for 20 minutes at 560 C. A pink color inidicates the presence of glyoxylic acid.
Acetic acid which was used for cholesterol
determina-tions gave a faint or negative reaction with this test. 6. Ferric chloride-sulfuric acid cholesterol reagent
(23): One g. of reagent grade FeCl3 6H2O (Coleman & Bell Co.) was dissolved in glacial acetic acid and
made up to 10.0 ml. in a volumetric flask. Five ml. of
this solution was diluted to 500 ml. with concentrated
suliuric acid (Baker and Adamson C.P. reagent). The resulting pale yellow solution was stored in a
separa-tory funnel plugged with a drying tube containing
an-hydrous calcium sulfate (Drierite) to prevent absorption of moisture from the air. Reagent kept in this way over
one month gave the same results as fresh reagent.
Procedure
1. Elcctrophoresis: Electrophoretic separation was
car-ried out on eight 2.9X30.0 cm. Whatman 3 MM filter
paper strips in a cell of the hanging strip type (27). To minimize variations in the final color caused by
ma-terial in the paper, all strips used in each determination were cut from the same sheet of filter paper and care
was taken to keep the strips clean and free from
per-spiration. After the strips had been wet with buffer and allowed to drain for 151 minutes, 0.02 ml. of serum was
applied in a streak at the apex of each strip with a
mi-cropipette (Microchemical Specialties Co., Berkeley, California), and a potential of 220 volts (14-17 m.a.)
was applied for two hours. The strips wsere then spread flat to avoid shifts in the bands during drying, and dried with a current of air at room temperature. Each serum was run in duplicate along with an additional sample for
staining.
2. Staiitintg: Those strips which were to be used to
locate the lipoprotein bands were dried horizontally for 10 minutes in an oven at 1100 C., stained for one hour
in Sudan black staining solution, rinsed for a few
min-utes in tap water, blotted, and again dried. One strip could be used for the location ofthe lipoprotein bands of
two different sera by applying two 0.01 ml. samples to a
strip witha 2X 120mm. slit cutdown its ceniter (Figure
1).
3. Elution: The apparatus used for eluting cholesterol from the strips is shown in Figure 2. Athermostatically controlled heating block maintains the solvent in the tubes at a steady boil with the vapors condensing on air cooled stainless steel condensers or "cold finger" con-densers constructed from conical centrifuge tubes.
Pa-per segments attached to the condensers are exposed to
.: .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. of
FIG. 1. SLOTTED PAPER STRIP SHOWING TYPICAL ALPHA AND BETA LIPOPROTEIN BANDS OF
Two SERA STAINED WITH SUDAN BLACK
The lines represent the point for division of the bands on corresponding unstained strips.
The carets show the point of serum application.
.R.
MEASUREMENT OF ALPHA AND BETA LIPOPROTEIN CHOLESTEROL
00 0 0 00
FAN
I
FT.
WATER COOLED
-r"COLD FINGER"CONDENSER
I.
DIA. |
25mm 35mm
12.5mm
DIA.
I
9mm 45mmAIR CONDENSER
1ERMOSTAT
FENWAL No.17000
FIG. 2. APPARATUS USED FOR ELUTION OF SERUM CHOLESTEROL FROM PAPER
STRIPS
a constant flow of fresh solvent, thus accomplishing an elution similar in principle to a Soxhlet extraction. The thermostat on the block is adjusted so as to maintain 4 ml. of water in an elution tube at 72 to 730 C. This provides for rapid boiling without excessive loss of
sol-vent during the elution. The tops of the elution tubes
are ground at a bevel to provide a close fit to the metal condensers.
The lipid patterns on the stained strips were marked off into two 3 cm. segments which best separated the alpha and beta fractions and which together included the entire lipid pattern (Figure 1). Identical segments were thenmarkedoff onthe unstained strips by aligning them with the corresponding stained pattern at the point of
serum application. These unstained segments were cut
out, folded with tweezersin one directionto four or five
thicknesses and forced into the wire loops on the
con-densers. A 3 cm. segment cut from the cathode side of
a strip was used as a blank. Four ml. of
chloroform-methanol solvent was added to each elution tube along with an alumina boiling chip, and the tubes were placed
inthe heating blockand allowed to come to aboil.. The
condensers were thenplaced inthe tubes and adjusted so
that the shafts did not touch the walls of the elution tubes, thus preventing the refluxing solvent from being
drained off the condensers onto the walls of the tubes without passing through the paper segments. The
seg-ments were eluted for two hours and the tubes were al-lowed to stand overnight.
4. Determination of cholesterol: The cholesterol in the eluate was determined by a modification of the
method of Zlatkis, Zak, and Boyle (23). After the con-densers were removed from the tubes and a fresh
boil-ing stone added, the solventwas again brought toa boil on the block. A cardboard cover with holes for the
openings of the tubes was placed over the apparatus to
help the evaporation by warming the tubes and prevent-ing condensation of vapor on their walls. The cover
was constructed so as to enclose the entire space above the heating block up to the top of the tubes. After the solvent had been allowed to evaporate for one hour the cover was removed, 1.00 ml. of glacial acetic acid
was added to the dry residue in each tube, and the tubes were replaced in the heated block for 15 minutes to in-sure solution of the residue. At this point two stand-ards were prepared by pipetting 0.02 ml. of the standard cholesterol solution into each of two elution tubes and adding 0.98 ml. of glacial acetic acid. A volume of 1.00 ml. of acetic acid was added to an elution tube for use as a reagent blank. After the tubes had cooled, 0.65 ml. STAINLESS STEEL
AIR CONDENSER
THOMAS A. LANGAN, E. DURRUM, AND WILLIAM P. JENCKS
of ferric chloride-sulfuric acid cholesterol reagent was added to each tube from a 10 ml. burette, allowing it to run slowly down the wall of the tube from a pointabout 3 cm. above the level of the acetic acid so as to form a layer under the acetic acid. Immediately after adding the reagent each tube was struck sharply with the fingers
to mix the reagents and placed in a 56° ± 10 C. water bath for 20 (17 to 23) minutes. Samples containing
cholesterol developeda brownish purplecolor, while elu-tion blanks were pale yellow. The solutions were allowed to cool toroom temperature, transferred tomatched 10X
75 mm.roundcuvettes, centrifugedbriefly to remove
bub-bles, and readwithinone hour ina Coleman Junior
Spec-trophotometer at 560 nm.. In transferring the viscous samples to the cuvettes, the elution tubeswereallowed to drain while supported in an inverted position by 1 x110 mm. glass rods placed in the cuvettes. The optical den-sity developed in the standard tubes showed a range of variation of 10 per cent over several months.
For the determination of eluted cholesterol by the
Sperry and Webb modification of the
Schoenheimer-Sperry procedure (28) it was necessary to combine
elu-ates from two strips. The evaporation was carried out until about 1 ml. of solvent remained in each tube, and
duplicate eluates were then combined, rinsing three times
with 1 ml., 0.5 ml., and 0.5 ml. of chloroform-methanol. The combined eluates were evaporated to dryness, and the residue extracted three times with 1 ml., 0.75 ml., and 0.75 ml. ofethanol-ether (3:1), transferring the ex-tracts to a 15 ml. conical centrifuge tube. Hydrolysis,
digitonin precipitation and color development were then
carriedout in the usual manner.
RESULTS
Color development and elution
Determination of the total cholesterol of whole
serum with the ferric chloride-sulfuric acid rea-gent without extraction, hydrolysis or precipita-tion (23) has in ourhands given resultswhich do
not consistently agree with those obtained with the Schoenheimer-Sperry method, in confirmation of the report by Best, Van Loon, Wathem, and
Seger (24). Use of acetic acid which had been refluxed over chromic acid and redistilled twice
(29) did not improve the results.
However, when the reaction was carried out on
chloroform-methanol extracts of serum which had
been electrophoretically separated on paper strips,
andthe temperature duringcolordevelopment was controlled byuse of a 560 water bath, satisfactory results were obtained.3
3After this work was completed, Zak, and his
co-workers published a method for the determination of total serum cholesterol with the ferric chloride-sulfuric
TABLE I
Colordevelopment with wholeserumand eluted serum using aceticacidof varying degrees of purity *
NetO.D.at560m/A
Acetic acid
Puri-fied Reagent U.S. P.
H2SO4-FeCl3 Wholeserum .379 .407 .433 Reagent Eluted serum .366 .375 .360
H2SO4 only Whole serum .097 .126 .144 Eluted serum .068 .055 .062
Glyoxylic acid reaction 0 + + + +
*Purified acetic acid was refluxed over chromic acid and
redistilled. Eluted samples are from serum streaked and driedonfilter paper. Colordevelopment was carried out asdescribed in the text.
Table I shows the influence of impurities in the acetic acid on the amount of color developed
in the determination of cholesterol on whole se-rum and on chloroform-methanol extracts of se-rum streaks dried on paper. It may be seen that with lots of acetic acid giving increasingly strong
glyoxylic acid reactions there is an increase in the
amount of color developed with whole serum, but
600-'0
z
.400-0. 0
200
20 40 60 80
j"gCHOLESTEROL
FIG. 3. STANDARD CURVE FOR PURE CHOLESTEROL acid reagent on alcohol-acetone extracts of sera which shows satisfactory agreement with the
Kingsley-Schaf-fert procedure (30).
MEASUREMENT OF ALPHA AND BETA LIPOPROTEIN CHOLESTEROL TABLEII
Recovery ofaddedcholesterol
Found
after Recovery
Serum addition ofadded Re-cholesterol of 20pg. cholesterol covery
Serum Fraction pg. cholesterol pg. %
Pool I* Alpha 8.9 29.6 20.7 104
Beta 31.5 50.2 18.7 94
1149 Alpha 7.1 27.6 20.5 103
Beta 36.2 57.0 20.8 104
1221 Alpha 10.2 31.2 21.0 105
Beta 23.0 43.5 20.5 103
1579 Alpha 8.5 29.3 20.8 104
Beta 41.4 62.0 20.6 103
* Pooled serum from 10blood donors.
ml SERUM
FIG. 4. DETERMINATION OF CHOLESTEROL IN ELUATES FROM ALPHA AND BETA LIPOPROTEIN FRACTIONS OF
IN-CREASING AMOUNTS OF SERUM
that no such increase occurs with eluates of the same serum. If ferric chloride is omitted so that
the reaction is carried out under the conditions of the glyoxylic acid-tryptophane reaction, the same
results are obtained. The small amount of color
developed in all samples without ferric chloride is probably due to the reaction of small amounts
of oxidizing substances present in the reaction
mixture with cholesterol.
Figure 3 shows thatthecolor developed with
in-creasing amounts of pure cholesterol increases
linearly up to an optical density of at least 0.700
when measured in the Coleman Junior Spectro-photometer after 20 minutes color development
in the 560 bath. After this incubation the color
is stable foratleast onehour at room temperature
orfor 20 minutes at560.
Figure 4 shows that a linear response is also
obtained with increasing amounts of serum
car-ried through the complete process of
electropho-retic separation, elution, and color development.
The total cholesterol of this serum as determined
by the Schoenheimer-Sperry technique was 212 mg. percent,as comparedto 216 mg.percent ob-tained by addingthealpha and betavalues shown.
Thus the efficiency of separation and elution of
theserum is not dependenton the quantity of
ma-terial involved over a range of one-half to two
times theamountordinarily used.
Table II shows the recovery of added choles-terol, which was streaked over alpha and beta fractions of sera which had been previously
sepa-rated and dried. The quantitative recovery ob-tained is evidence against the presence in the se-rum ofinhibitors or other impurities affecting the color development dueto cholesterol.
A comparison of the results obtained with the complete separation, elution and color development procedure on six different normal and abnormal
sera with the results of Schoenheimer-Sperry de-terminations of total serum cholesterol on the
same serais shown in Table III. The sum of the values obtained for alpha and beta lipoprotein cholesterol agrees with the results of the Schoen-heimer-Sperry method within the sum of the
er-TABLEIII
Recovery of elutedcholesterol determined by theferricchloride andSchoenheimer-Sperry methods
Serum
1589 1590 1592 1593 1595 PoolII*
Schoen- heimer-Sperry
serum
cholesterol
mg.%
202 331 238 332 328 288
Ferric chloride
eluted cholesterol
(sum of alpha andbeta)
mg. %
192 326 258 337 330 261
Re-covery
95
99 110 102 101 91
Schoen-
heimer-Sperry
eluted cholesterol
mg.%
213 322 247 309 306 264
Re-covery
106 97 103 96 93 92 Average
recovery 100 98
*Pool of 5sera. 0
0:
v> tA
;&g. Cholesterolrecovered Alpha Beta
10.3 34.4
-0.1* -0.4*
-0.2* 0.4
-0.2* 0.3
Serum: Pool I from 10 blood donors.
Cholesterol applied (Schoenheimer-Sperry method): 42.4 ,ug.
Cholesterol recovered (Ferric chloride method,sum
of 1i hr. alpha and beta values): 44.7,g.
*Negativefiguresrepresentvalues lower thanthe blanks.
These differences are no greater than the error of the
determination.
rors of the two methods. To insure that the
ap-parently complete recovery was not an artifact
duetoacombination ofincomplete elution and
er-roneously highvalues with the ferric chloride
pro-cedure, the same sera were electrophoretically
separated, the entire lipid containing area was
eluted, and the combined eluates from two strips
were analyzed for cholesterol by the
Schoen-heimer-Sperry procedure. The last two columns of Table III show that these values agree
satis-factorily with those obtained with the ferric
chloride method.
Asafurthertestofthecompleteness ofthe
two-hour elution procedure, the alpha and beta
seg-ments oftwo stripswereeluted foratotal of
nine-teenhours, transferring thesegments tofresh
elu-tion tubes at intervals and determining the
cho-lesterol extracted during each period. As shown
in Table IV, no additional cholesterol was
re-covered after one and one-half hours of elution.
The recovery (sum of one and one-half hours
alpha and beta values vs. Schoenheimer-Sperry
total) was 105 per cent.
The blank values obtainedby elution of anarea
of the paper strip containing no protein could be
lowered somewhat by extraction of the strip with
fat solvents before use. However, since the net
values for eluted serum cholesterol were not
changed by suchaprocedure, itwasnotordinarily
carried out. The buffer salts in the dried paper,
which are elutedalong with the lipid material, do
notgivecolor oraffect the colorreaction.
Electrophoretic separation
Separation of the alpha and beta lipoprotein
fractions of serum was carried out simultaneously
on eight filter paper strips moistened with pH 8.6 barbital buffer in the cell previously described (27). Using 0.02 ml. of serum, a clear cut sepa-ration of alpha and beta lipoproteins as revealed by Sudan black staining was invariably obtained. Figure 1 shows a typical "marker" strip which
was used to locate the lipoprotein bands for each of the sera run in the same cell.
The point used for dividing the alpha and beta fractions was about 2 mm. ahead of the sharply de-fined front of the beta lipoprotein band; no evi-dence was obtained by staining for the presence of other lipoprotein fractions in practicably measur-able amounts. However, in certain pathological sera, the "beta lipoprotein" band moved ahead of the beta globulins as revealed by protein staining. Since all gradations of mobility between
alpha,
and beta globulin were observed for thislipopro-teinfraction inaseries ofsuch sera, while the mo-bility of alpha lipoproteins was remarkably con-stant, it seemed most reasonable to consider these
to be abnormal forms of beta lipoprotein rather than anotherfraction of alpha lipoprotein. No at-tempt was made to distinguish between
chylomi-crons and beta lipoprotein
adsorbed
at the appli-cationpoint; such a differentiation wouldpresum-ablybe of little importance in any case because of
the low cholesterol content of chylomicrons (1).
Under these conditions of electrophoretic
sepa-ration, the leading edge of the alpha lipoprotein band migrated 3 to 4 cm. from the origin. This relatively short migration distance was selected in order to minimize the possibility of error due to
"tailing" of alpha lipoprotein and the high blank values obtained if large amounts of paper are
eluted. As mentioned above, "tailing" of beta lipoproteins is of no consequence, since any beta "tail" is eluted with the band itself. The linear recovery of cholesterol obtained with electrophore-sis and elution of increasing amounts, of serum
(Figure 4) rules out the possibility of significant
adsorption of the alpha lipoprotein of the kind ordinarily observed, in which a fairly constant
amount ofmaterial is adsorbed on agiven area of paper regardless of the amount applied. The oc-currence of an appreciable amount of this type of
"tailing" would result in a displacement of the
alpha and beta lipoprotein lines so that the latter would intersect the ordinate above zero, and the former below zero, as found with albumin
"tail-TABLE IV
Elutionofcholesterolfromalphaand betalipoproteins
Elutionperiod
0 tolI hrs.
ljhrs. to 2 hrs. 2hrs.to 4 hrs.
MEASUREMENT OF ALPHA AND BETA LIPOPROTEIN CHOLESTEROL
UNDER N2
20
0
A0- a UNDER N
z> 2 ~~3 4
DAYS STANDING IN AIR
FIG. 5. DECOMPOSITION OF ALPHA AND BETA
Lipo-PROTEIN CHOLESTEROL ON PAPERSTRIPS STANDINGIN AIR
AT ROOM TEMPERATURE
ing" (31, 32). It is likely that albumin, passing
over the paper surface in front of the alpha
lipo-protein, covers up the available adsorptive sites on thepaper, thus "laying down a carpet" for the
globulin fractions.
Decomposition of cholesterol
The recovery of cholesterol from a series of
identical strips eluted at varying periods of time after electrophoretic separation is shown in
Fig-ure 5. It may be seen that after the strips had
stood in air at room temperature for four days there was a 55 per cent loss of cholesterol as
de-termined by theferric chloride procedure. Strips
which had been kept under nitrogen for the same
periodof time showedonly a 16percent loss,
sug-gesting that oxidation was responsible for the
de-composition. Inasimilarexperiment strips which
had been allowed to stand for seven days in air
showed a 42 per cent loss by the ferric chloride
method, and a 27 per cent loss by the
Schoen-heimer-Sperry procedure. In order to avoid this
typeofdecomposition, the dried strips wereeluted
within a few hours of the electrophoretic
separa-tion. No losses were observed on allowing the
eluted cholesterol to stand in solution overnight.
Oxidation of cholesterol under these conditions
is not unexpected in view of the results of
Berg-str6m and Wintersteiner (33) who observed a
rapid oxidation ofcolloidal cholesterol inwaterby
astream of airatelevatedtemperatures anda
sig-nificantrate of decompositionat37°.
Inview of theseobservations, it appeared neces-sary toexamine the possibility of losses of
choles-terol during the electrophoretic procedure itself.
Experiments in which pure cholesterol was
ap-pliedtostrips froma solution of acetic acid, dried,
the strips moistened with buffer, and the complete electrophoretic and elution procedure carried out,
did show recoveries which were 12 to 15 per cent
low. However, numerous experiments on serum,
including those reported in Table III, failed to
show any evidence of decomposition and no
dif-ference in cholesterol recovery was observed if
the electrophoretic procedure was carried out at
0to 10 C. inanattempt to decreaseany oxidation
that might be taking place by lowering the tem-perature. Table II, already referred to, shows
that pure cholesterol added to the area of the
alpha and beta bands is recovered completely. It appears that decomposition of cholesterol
does not readily take place in solution, but is
ap-preciable when exposed to air in the solid or
col-loidal state. It is possible that serum exerts a
specific protective effect because of the presence
of antioxidants in serum. It is generally known
that while lipoproteins are stable in serum for
several weeks at 50, theyundergo rapid oxidation
in the absence of serum as shown by increases in
electrophoretic mobility and decreases in
ultra-centrifugal flotation rates (34, 35).
As a further check onthe method, the ratios of
the alpha and beta lipoproteins of three pooled
sera were determined by both the
Schoenheimer-TABLEV
Comparison ofthe Schoenheimer-Sperry andferric chloride methods in thedeterminationof lipoprotein
cholesteroldistribution
%of total cholesterol in alpha lipoprotein fraction Schoenheimer- Ferric
Sperry chloride Serum method method
Pool III* 23.4 22.9
21.4 21.9
Pool IV* 20.5 19.7
19.4 19.4
Pool
V* 20.4 19.918.3 18.5
*Pooledserumfrom10blooddonors.
TABLE VI
Reproducibility of lipoprotein cholesterol distribution and total cholesterolinfiveseparatedeterminations
onsixnormaland abnormalsera
Serum 1149 1209 1221 1493 1579 Pool I* Experiment No. 1 2 3 4 5 Average 1 2 3 4 5 Average 1 2 3 4 5 Average 1 2 3 4 5 Average 1 2 3 4 5 Average 1 2 3 4 5 Average *Pooled
serumfrom 10 blooc
%O Alpha lipoprotein cholesterol 19.0 18.6 17.8 17.3 19.6 18.5 16.0 15.7 16.8 16.6 16.7 16.4 30.4 30.7 31.6 32.5 31.8 31.4 14.1 13.8 13.6 14.3 15.4 14.2 17.5 16.9 18.6 16.2 17.9 17.4 24.7 22.0 24.4 25.0 26.9 24.6 Total cholesterol (sumofalpha
andbeta) mng.% 240 218 224 230 235 229 315 303 307 327 322 315 191 170 175 191 197 185 336 312 318 334 333 327 280 252 269 274 283 272 228 211 218 222 230 222 d donors.
Sperryand ferric chloride methods. Because of the
comparativelylowsensitivity ofthe
Schoenheimer-Sperry method,it was necessary toelute segments
from strips on which 0.04 ml. of serum had been run, and to combine the eluates from four alpha
segments and from two beta segments for the determination of cholesterol inthe respective frac-tions. The results of such an experiment, shown
in Table V, indicate that closely comparable
val-ues for the distribution of cholesterol between the
alpha and beta lipoproteins are obtained by both
methods and that no combination of errors in
the ferric chloride method is giving rise to in-correct values for alpha and beta lipoprotein cho-lesterol while giving satisfactory agreement be-tween the two methods for total cholesterol (Table III).
Reproducibility
The results of a study of the reproducibility of the method under conditions of routine use are
shown in Table VI. The complete separation,
elution and color development procedure was car-ried out in duplicate on each of six sera in five separate experiments over a period of nine days. The widest range of variation observed was from 22.0to 26.9 per cent for the fraction of total cho-lesterol found in alpha lipoprotein (24.4 to 26.9 per cent if one low value obtained for this serum
is omitted), and from 252 to 283 mg. per cent for the total cholesterol (sum of alpha and beta
frac-tions).
DISCUSSION
The available methods for determination of lipoprotein concentration are dependent on
re-fractive index increment, dye binding capacity,
or the content of some chemically defined
ma-terial such as phospholipid or cholesterol in the lipoprotein molecule. Of these methods, the de-termination of cholesterol has the advantage of
being based on a specific chemical reaction of a
substance of known importance in atherosclerosis, while the refractive index increment is dependent
on the concentration of both protein and lipids. The measurement of uptake of lipid dyes by lipo-protein fractions, which is dependent on solubility interrelationships between dye, solvent and lipid,
does not appear to have as firm a basis as either
ofthese methods (36). It should be keptinmind that lipoprotein concentrations expressed in these various units are not directly comparable because of the different composition of different normal and abnormal lipoproteins.
The wide application of some of the methods
of serum lipoprotein separation and estimation
has been restrictedby thetime, technique, amount
MEASUREMENT OF ALPHA AND BETA LIPOPROTEIN CHOLESTEROL
eluates from electrophoretically separated serum
has the advantages of being relatively simple and of requiring a minimum of serum and
equip-ment. The time required and the number of sera
which can be conveniently analyzed together are
approximately the same as for the
Schoenheimer-Sperry determination of total cholesterol, while both total cholesterol and its lipoprotein distribu-tion are determined with the electrophoretic procedure.
SUMMARY
1. A method is described for the determination of the alpha and beta lipoprotein cholesterol of 0.02 ml. of serum separated by electrophoresis on filter paper.
2. Under the conditions described, the following criteria are satisfied:
a. A linear increase in color for increasing amounts of pure cholesterol and eluted alpha and beta lipoprotein cholesterol.
b. Agreement, within the errors of the two methods, of total cholesterol and of per cent alpha lipoprotein cholesterol as determined by the method described and by the Sperry and Webb modification of the Schoenheimer-Sperry pro-cedure.
c. Complete recovery of eluted lipoprotein cho-lesterol.
d. Clear cut separation of alpha and beta lipo-proteins.
e. Satisfactory reproducibility of the results in repeated determinations on the same sera.
3. Dry cholesterol on filter paper strips
ex-posed to air at room temperature undergoes
de-composition.
4. The method is comparable, in terms of time andnumber ofseraanalyzed,tothe Schoenheimer-Sperry technique.
ACKNOWLEDGMENTS
The authors are greatly indebted to Miss Marion Hyatt for help with the Schoenheimer-Sperry choles-terol determinations, and to the Department of
Bio-physical Instrumentation for construction of the elution
apparatus.
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